內生性大麻素Anandamide誘發肝癌細胞週期停滯和細胞凋亡及其相關轉錄因子STAT3和NF-κB之調控作用

Anandamide Induces G1 Cell Cycle Arrest and Apoptosis in Human Hepatoma Cells through the Regulation of Transcription Factors STAT3 and NF-κB

楊軍建(Chun-Chien Yang)

指導教授 ： 邱慧芬

高雄醫學大學/醫學院/藥理學科研究所/碩士(2012年)

摘要

Anandamide (AEA)，是第一個被分離出來的內生性大麻素，已被許多報告指出對數種癌症細胞株具有抗腫瘤的活性，其中包括乳癌、前列腺癌、子宮頸癌、淋巴癌等。
肝癌是亞洲地區癌症死亡的主要原因之一，而AEA對肝癌的的毒殺能力、抗癌機轉以及相關之細胞週期及程式死亡調控作用及對轉錄因子的影響則是目前尚未被清楚解析，因此亦是我們從事本研究之動機。
本篇論文首要研究AEA對於人類肝癌細胞的毒殺作用，再進一步探討AEA 所引起之細胞週期調控、細胞凋亡誘導作用和對細胞核中之轉錄因子包括Signal transducers and activators of transcription factor 3 (STAT3)和Nuclear factor-κB (NF-κB)兩者相關調控之機轉，以期了解AEA抗肝癌活性上分子機轉。
本研究以XTT 和 methylene blue 兩種方法，研究不同濃度 的AEA (1、10、20、30、50 μM)對於人類肝癌細胞 (HepG2和2.2.15)、人類肺癌細胞(A549) 、人類乳癌細胞(MCF-7) 人類大腸癌細胞(HT-29)及大白鼠神經膠質瘤(C6)之毒殺作用，結果發現兩種毒殺方法皆顯示細胞生長之調節呈現劑量依存性(dose-dependent)的結果。
以Comet assay 之分析觀察DNA損害程度，發現以50 μM AEA藥物作用3到6小時後產生明顯彗星拖尾現象。AEA造成的DNA受損可能引起後續的凋亡及細胞週期停滯情形。
之後的結果顯示AEA引起的凋亡情形伴隨著以下三種事件的發生：(A)活性氧化物(reactive oxygen species, ROS)的測定結果中發現ROS之產生隨AEA劑量增加而上升，因此使得細胞內之氧化壓力增加，此現象趨向後續並利於走向細胞程式死亡。(B) 以DiOC6染色發現以AEA處理會引起粒線體膜電位受損。(C) 粒線體中cytochrome C蛋白質的減少及細胞質中cytochrome C蛋白質的增加。
以流式細胞儀 (flow cytomety )測定細胞週期變化，加入不同濃度之AEA (1.0-50 μM)後會造成細胞週期停滯於G1期且此效應呈現濃度依賴性的反應，進一步利用西方墨點法以偵測AEA對於G1期中相關訊息蛋白質表現之影響，發現細胞週期素cyclin D1及其依賴酵素 CDK4 和Cyclin E/ CDK2以及轉錄因子E2F1和磷酸化Rb蛋白質均有呈現表現量下降的現象。相反的，負責抑制細胞週期進行的相關蛋白質如 Rb, p21、p27及p53則是有表現量增加的情形，以上AEA之諸多作用進而導致細胞週期停滯於G1期的結果。

在AEA細胞凋亡誘導作用之測定中，以不同濃度(1~50 μM)的AEA作用於肝癌細胞48小時後，再以Annexin V / propidium iodide (PI) 作雙重染色，接著以流式細胞儀測定細胞程式死亡之現象，結果發現AEA引起細胞凋亡的數目會隨著濃度的增加而呈現依存性的現象(dose dependent response)。另外再利用西方墨點分析法中證實AEA 促進細胞凋亡之訊息蛋白如Fas、Fas ligand、Bax、Caspase 3/8/9等之表現，並同時會減少抗凋亡作用之蛋白質Bcl-2等的表現，我們的結果支持AEA處理會活化內在及外在的凋亡路徑。
STAT3 及NF-κB在各種癌症中被證明是會持續活化的兩種癌化轉錄因子，本研究藉著證明AEA會抑制STAT3及NF-κB與相關DNA的接合能力及進核的現象，說明AEA 會抑制STAT3及NF-κB在肝癌細胞中之活性。
總結本研究之結果，我們發現內生性大麻素 AEA會經由細胞週期G1期停滯作用及引起細胞凋亡之誘導進而抑制肝癌細胞之增生，而此現象之機轉可能經由調控細胞核之中轉錄因子STAT3及NF-κB的活性而成。

關鍵字

內生性大麻素

並列摘要

Anandamide (N-arachidonoylethanolamide, AEA), an endogenous cannabinoid (endocannabinoid), possesses a wide spectrum of physiological and pathological functions. Recent evidence indicates that endocannabinoids possess anti-tumor effects in a number of different tumor cells, including breast, prostate, cervical carcinoma and lymphoma tumor cells.
Hepatocellular carcinoma (HCC) is major of cancer deaths in Asia. However, little is known about the exact anti-cancer molecular mechanism of AEA in HCC. The purpose of this study was to elucidate the effects of AEA on the modulation of cytotoxic effects and related action mechanisms involved in cell cycle arrest, apoptotic induction and transcription factor modulation in HCC.
We found that the anti-proliferative effects of AEA in different cancer cell lines including human hepatoma (HepG2 and 2.2.15), human lung adenocarcinoma cells (A549), human breast cancer cells (MCF-7), human colon cancer (HT-29) and rat glioma cells (C6) acted in a concentration-depend manner. In comet assay analysis, AEA induced DNA damage after 3 to 6 hours of treatment in a dose-dependent phenomenon. The DNA damage in AEA-mediated anti-proliferation was associated with cell cycle arrest and apoptotic induction.
Further investigations revealed that AEA-mediated apoptotic induction was accompanied by the following three events: (A) generation of reactive oxygen species (ROS) (measured by DCHF staining); (B) dissipation of mitochondrial membrane potential (assayed by DiOC6 labeling); and (C) a decrease of mitochondrial cytochrome c and an increase of mitochondrial cytochrome c.
Cell cycle G1 arrest was observed in HepG2 cells with various concentrations of AEA in a dose-dependent manner. In Western blot analysis, AEA up-regulated protein signal expressions of p27kip1, p21waf1, Rb and p53, and down-regulated the G1 phase related cyclin D1, E and CDK 2/4 expressions. In addition, levels of transcription factors E2F1 and phospho-Rbser780 declined in AEA-treated cells.
Flow cytometry and annexin V labeling assay indicated that AEA increased apoptotic cells in a dose-dependent manner. AEA up-regulated expressions of apoptotic signal proteins including Fas, Fas-ligand, Bax and caspase 3, 8, and 9, and down-regulated the expression of anti-apoptotic Bcl-2. Our findings support that AEA may activate intrinsic and extrinsic apoptosis pathways.
Signal transducers and activators of transcription factor 3 (STAT3) and nuclear factor-κB (NF-κB) are important transcription factors in tumorigenesis. We demonstrated that AEA inhibited constitutive activation of STAT3 and NF-κB. The inhibition corresponded with and with the suppression of DNA binding and nuclear translocation of STAT3 and NF-κB.
In summary, our results showed that AEA exerted an anti-proliferative effect on human hepatoma cells by inducing cell apoptosis and cell cycle G1 arrest. The mechanism may be through the inhibition of STAT3 and NF-κB activities.

並列關鍵字

Anandamide ； STAT3 ； NF-κB

參考文獻

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